ORIGINAL ARTICLE

Neuronal morphology in the African elephant(Loxodonta africana) neocortex

Bob Jacobs • Jessica Lubs • Markus Hannan •

Kaeley Anderson • Camilla Butti • Chet C. Sherwood •

Patrick R. Hof • Paul R. Manger

Received: 14 August 2010 / Accepted: 15 October 2010 / Published online: 16 November 2010

� Springer-Verlag 2010

Abstract Virtually nothing is known about the mor-

phology of cortical neurons in the elephant. To this end, the

current study provides the first documentation of neuronal

morphology in frontal and occipital regions of the African

elephant (Loxodonta africana). Cortical tissue from the

perfusion-fixed brains of two free-ranging African ele-

phants was stained with a modified Golgi technique.

Neurons of different types (N = 75), with a focus on

superficial (i.e., layers II–III) pyramidal neurons, were

quantified on a computer-assisted microscopy system using

Neurolucida software. Qualitatively, elephant neocortex

exhibited large, complex spiny neurons, many of which

differed in morphology/orientation from typical primate

and rodent pyramidal neurons. Elephant cortex exhibited a

V-shaped arrangement of bifurcating apical dendritic

bundles. Quantitatively, the dendrites of superficial pyra-

midal neurons in elephant frontal cortex were more com-

plex than in occipital cortex. In comparison to human

supragranular pyramidal neurons, elephant superficial

pyramidal neurons exhibited similar overall basilar den-

dritic length, but the dendritic segments tended to be longer

in the elephant with less intricate branching. Finally, ele-

phant aspiny interneurons appeared to be morphologically

consistent with other eutherian mammals. The current

results thus elaborate on the evolutionary roots of Afro-

therian brain organization and highlight unique aspects of

neural architecture in elephants.

Keywords Dendrite � Morphometry � Dendritic spine �Golgi method � Brain evolution

Introduction

In recent years, elephants have received considerable

attention because of their complex social structure and

cognitive abilities (Poole and Moss 2008; Hart and Hart, in

press). Nevertheless, despite elephants’ status as the largest

living terrestrial mammal, very little is known about the

brain of this species. Prior to 2001, only a few original

articles had specifically focused on the elephant central

nervous system (Cozzi et al. 2001). Although the gross

anatomy of the *5 kg adult elephant brain has been

investigated in the last decade (Kupsky et al. 2001; Hakeem

et al. 2005, 2009; Shoshani et al. 2006; Manger et al. 2010),

virtually nothing is known about the microstructural mor-

phology of its neurons. Indeed, until recently, the only

apparent insight into elephant cortical neuromorphology

was a single camera lucida drawing from an Indian elephant

(Elephas maximus; Barasa and Shochatovitz 1961). Rea-

sons for this poverty of data are many (Cozzi et al. 2001),

but include a propensity for neuroscientists to focus on the

rodent and primate species commonly used in biomedical

B. Jacobs (&) � J. Lubs � M. Hannan � K. Anderson

Laboratory of Quantitative Neuromorphology, Psychology,

The Colorado College, 14 E. Cache La Poudre,

Colorado Springs, CO 80903, USA

e-mail: BJacobs@ColoradoCollege.edu

C. Butti � P. R. Hof

Department of Neuroscience and Friedman Brain Institute,

Mount Sinai School of Medicine, One Gustave L. Levy Place,

New York, NY 10029, USA

C. C. Sherwood

Department of Anthropology, The George Washington

University, 2110 G Street, NW, Washington, DC 20052, USA

P. R. Manger

Faculty of Health Sciences, School of Anatomical Sciences,

University of the Witwatersrand, 7 York Road, Parktown,

Johannesburg 2193, South Africa

123

Brain Struct Funct (2011) 215:273–298

DOI 10.1007/s00429-010-0288-3

research (Manger et al. 2008), and a lack of well-preserved

elephant brain tissue suitable for histological analysis. This

latter issue was recently addressed by Manger et al. (2009),

who were able to fix the brains of wild African elephants

(Loxodonta africana) by postmortem, carotid cannulation-

perfusion. The quality of this tissue facilitated the present

investigation, which qualitatively and quantitatively

explores, for the first time, the neuromorphology of frontal

and occipital cortices in the African elephant.

Elephants belong to the order Proboscidea, which

emerged approximately 60 million years ago from within

the eutherian superorder Afrotheria (Sukumar 2003;

Shoshani and Tassy 2005; Gheerbrant and Tassy 2009;

Fig. 1). Extant afrotherian species (e.g., elephants, mana-

tees, dugongs, hyraxes, aardvarks, golden moles, tenrecs,

and elephant shrews) exhibit significant diversity in terms of

body and brain mass, neural organization, and life history

(Gravett et al. 2009; Pieters et al. 2010). Because the afro-

therian clade represents a major adaptive radiation within

Eutheria, it is in a position to provide insight into mamma-

lian brain evolution, with current evidence suggesting that

Afrotheria contains many neural plesiomorphies in com-

parison with other placental mammal species (Sherwood

et al. 2009). Thus, examination of neuronal morphology in

elephants is essential for understanding broader phyloge-

netic patterns of neuromorphological evolution in Afrothe-

rians and provides data concerning the scaling of neuronal

somatodendritic geometry with increasing body and brain

size (Tower 1954; Haug 1987; Hart et al. 2008).

Both cognitive and sociobehavioral investigations of

elephants reinforce the view that such a large brain is

associated with extensive abilities (Roth and Dicke 2005;

Hart and Hart, in press). The cognitive repertoire of ele-

phants includes elementary tool construction/usage (Hart

et al. 2001), impressive spatial and temporal memory

(Markowitz et al. 1975; Hart et al. 2008), creative problem

solving skills (Bates et al. 2008b), the ability to classify

human ethnic groups by odor and garment color (Bates

et al. 2007a), and the ability to locate out-of-site family

members by smell (Bates et al. 2007b). Social interactions

in elephants are underscored by several documented abil-

ities: ultra-low frequency sound communication over both

local and long distances (Poole 1999; Garstang 2004; Soltis

2009), vocal learning (Poole et al. 2005), long-term care of

their young (Bates et al. 2008b), individual identification of

conspecifics (Bates et al. 2007b), and targeted helping

behavior (Hart et al. 2008). These abilities suggest ele-

phants may be capable of empathy (Bates et al. 2008a, b)

and self-recognition, including theory of mind (Plotnik

et al. 2006), all of which crucially contribute to their

complex social networks (McComb et al. 2000, 2001).

Unfortunately, besides the recent documentation of von

Economo neurons in the anterior insular cortex of elephants

(Hakeem et al. 2009), one can only extrapolate the neural

foundations for these cognitively demanding abilities. For

example, based on the low density of neurons in their

cerebral cortex, it has been suggested that the elephants’

cognitive faculties may be concentrated in long-term pro-

cessing (Hart and Hart 2007; Hart et al. 2008), although fine

sensorimotor integration is certainly required to guide the

trunk (Hoffmann et al. 2004). Apart from such speculation,

there are currently no data that allow for direct examination

of the computational biophysics of elephant cortical neu-

rons. However, in the prototherian echidna, whose ancestry

is close to the root of the mammalian phylogenetic tree

(Fig. 1), a large proportion of pyramidal neurons contain so-

called ‘‘atypical’’ features, such as bifurcated apical den-

drites, inverted somata, poorly developed basilar skirts, and

a lack of terminal bouquets in layer I (Hassiotis et al. 2003,

2005). This suggests that there may be significant phyloge-

netic diversity in the morphology of spiny projection neu-

rons of the cerebral cortex. As such, based on its afrotherian

origin, we expect a variety of spiny neuron morphologies in

the elephant that differs from the euarchontoglire branch of

Fig. 1 Taxonomy of the elephant (Loxodonta africana) and a

sampling of related species mentioned in the present paper. During

the late Cenozoic era, Eutheria (placental mammals) diverged into

two phylogenetic groups: Atlantogenata and Boreoeutheria (Wildman

et al. 2007). Atlantogenata subsequently diverged into two sister

clades, Xenarthra (e.g., sloths, anteaters, and armadillos) and

Afrotheria, whereas Boroeutheria diverged into Laurasiatheria (e.g.,

cetaceans, carnivores, and ungulates) and Euarchontoglires (e.g.,

primates and rodents)

274 Brain Struct Funct (2011) 215:273–298

123

placental mammals, mostly typified by anthropoid primate

and murid rodent models, and thus appear ‘‘atypical’’. In

contrast, the morphological classes for aspiny interneurons

are more uniform across eutherian mammals (Hof et al.

1996, 1999; DeFelipe et al. 2002; Hassiotis and Ashwell

2003; Hof and Sherwood 2005). Thus, we expect aspiny

interneurons in elephants to resemble more closely the types

that are common in the mammalian cerebral cortex.

By extension, neuromorphological diversity in the ele-

phant implies a diverse cytoarchitecture, which would

contradict the long-held, but problematic claim that large

brains—such as in cetaceans—lack histological complexity

(Kesarev 1975; Glezer et al. 1988). In fact, recent studies on

cetacean cerebral cortices have revealed a complex cytoar-

chitecture, comparable to that observed in many mammals,

including primates (Hof et al. 2005; Hof and Van der Gucht

2007; Butti et al. 2009; Butti and Hof 2010). The present

study examines such neuromorphological diversity by

exploring regional variation in neuron morphologies across

all layers of the elephant cortex and by examining quanti-

tative differences in superficial (i.e., layers II–III) pyramidal

neurons between frontal and occipital cortical regions. As is

the case with human supragranular pyramidal neurons, these

superficial pyramidal neurons also tended to stain clearly

with the rapid Golgi technique (Jacobs et al. 2001). Both

functional (Foxe and Simpson 2002) and morphological

data in primates (Elston and Rosa 1997; Jacobs et al. 2001)

suggest regional variation in cortical processing, generally

with successively more complex processing as one moves

rostrally. Greater processing demands on neurons appear to

be reflected in the increasing complexity of their dendritic

arrays (Elston 2003, 2007; Jacobs and Scheibel 2002).

Although it is not possible to know which functional cortical

areas are contained in the frontal and occipital samples in the

present study, examination of dendritic complexity, as

revealed by superficial pyramidal neurons, may indicate

regional variation of integrative functions in the elephant.

Materials and methods

Specimens

Two (LA1 and LA3; Fig. 2a), free ranging, solitary male

African elephants (Loxodonta Africana; approximate age

Fig. 2 a Photograph of elephant LA1; b superior and c inferior views

of the elephant brain illustrating the relative position of sampled

tissue blocks (shaded areas) for the occipital and frontal regions,

respectively. Note that, in c, the right hemisphere’s olfactory bulb has

been removed in order to view the orbital gyri and facilitate removal

of the tissue block. Scale bar 5 cm

c

Brain Struct Funct (2011) 215:273–298 275

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20–30 years) scheduled for population management culling

were humanely killed as described in Manger et al. (2009).

In situ perfusion-fixation of the brains was conducted by

removal of the head, flushing of the cranium with cold

saline, and intra-carotid perfusion with 4% paraformalde-

hyde in 0.1 M phosphate buffer, resulting in relatively

short autolysis times (LA1: 160 min; LA3: 110 min). The

brains were then removed from the skull, placed in the

same cold fixative, weighed (LA1: 5,145 g, LA3: 4,835 g),

and stored in 4% paraformaldehyde in 0.1 M phosphate

buffer for 72 h. Small tissue blocks containing the cortical

regions of interest were removed and stored in 0.1%

sodium azide in 0.1 M phosphate buffer saline at 4�C for

8 months before staining. The present research protocol

was approved by the Colorado College Institutional

Review Board (#H94-004) and by the University of the

Witwatersrand Animal Ethics Committee (Manger et al.

2009).

Tissue selection

Frontal and occipital tissue blocks (1–2 cm, perpendicular

to the long axis of the sampled gyri) were removed from

the right hemisphere. Because no cytoarchitectural criteria

exist in the elephant for identification of these cortical

regions, localization was based solely on gross examination

of elephant brains (Haug 1970; Hakeem et al. 2005;

Shoshani et al. 2006) and on reports from other species

(e.g., giraffes: Badlangana et al. 2007; cetaceans: Hof and

Van der Gucht 2007). Occipital tissue was removed from

the parieto-occipital contour close to the midline, approx-

imately 3–5 cm immediately anterior to the cerebellum,

although it should be noted that the elephant occipital lobe

is not as clearly demarcated as in humans (Shoshani et al.

2006; Fig. 2b). Frontal tissue was removed from the

anterior portion of what appeared to be the orbital gyri,

3–5 cm from the midline, in a position superior to the

anterior third of the olfactory bulb (Fig. 2c).

Tissue blocks were trimmed to 3–5 mm in thickness,

coded to prevent experimenter bias, and processed by a

modified rapid Golgi technique (Scheibel and Scheibel

1978b). To be consistent with previous studies (Jacobs

et al. 1997, 2001), processed tissue was serially sectioned

at 120 lm with a vibratome. Tissue blocks adjacent to

those removed for Golgi analysis were examined with a

routine Nissl stain to establish laminar and cortical depth.

Thirty depth measurements for each layer were averaged

across 12 sections from each cortical region.

Neuron selection and quantification

Analyses qualitatively and quantitatively characterized

elephant neocortical neurons (N = 75), with a particular

focus on superficial (i.e., layers II–III) pyramidal neurons

(mean soma depth = 794 ± 131 lm) in the two cortical

regions. Because the Golgi impregnation was not uniform,

more neurons appropriate for quantification were sampled

from LA1 (n = 46) than from LA3 (n = 29). A total of 40

superficial pyramidal neurons were traced in the occipital

cortex (15 from LA1; 5 from LA3) and in the frontal cortex

(20 from LA1); of these, 16 had relatively complete apical

dendrites, which were analyzed only descriptively.

Although pooling data across animals was not an optimal

solution, an ANOVA comparing occipital total dendritic

length in superficial neurons between LA3 and LA1 found

no significant difference, suggesting this as an acceptable

compromise for subsequent analyses. A broader sampling

of neuron morphology resulted in 16 additional, deep layer

III to layer VI pyramidal neurons being traced in occipital

cortex (10 from LA1; 6 from LA3), and 19 in frontal cortex

(1 from LA1; 18 from LA3).

Previously established selection criteria for human su-

pragranular pyramidal neurons (Jacobs et al. 1997, 2001;

Anderson et al. 2009) were adapted in the present study.

Briefly, selected neurons were required to be relatively

isolated and unobscured, to appear fully impregnated with

a soma roughly centered within the 120 lm-thick section,

and to have as complete dendritic arbors as possible. In

order to create a relatively homogeneous cell population

for superficial pyramidal neurons, a running average of

soma depth from the pial surface was maintained and cells

were chosen to preserve similar averages across both cor-

tical areas.

Cells were quantified along x-, y-, and z-coordinates

using an Olympus BH-2 microscope under a Planachromat

409 (NA = 0.70) dry objective interfaced with a Neu-

rolucida software system (MBF Bioscience, Williston, VT,

USA). The system utilized a MicroFire Digital CCD 2-

Megapixel camera (Optronics, Goleta, CA, USA), which

was mounted on a trinocular head. The camera image was

viewed on a Dell E248WFP 24-inch LCD monitor with

1,920 9 1,200 resolution. First, the soma was traced at its

widest point in the two-dimensional plane in order to

provide an estimate of its cross-sectional area. Dendrites

were then traced somatofugally in their entirety, account-

ing for dendritic diameter and quantifying all visible

spines, regardless of spine type or differences in spine

length. Dendritic processes were not followed into adjacent

sections; as such, broken tips and unclear terminations

were identified as incomplete endings.

Neurons were traced by three observers (BJ, JL, and

KA). Intrarater reliability was assessed by having each

rater trace the same dendritic branch, with somata and

spines, 10 times. Tracings varied little, as indicated by

the relatively low coefficient of variation for soma size

(2.8%), total dendritic length (TDL 2.8%), and dendritic

276 Brain Struct Funct (2011) 215:273–298

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spine number (DSN 3.9%). Intrarater reliability was also

tested with a split plot design (a = 0.05), which revealed

no significant difference within tracers between the first

five tracings and the last five tracings of a dendritic

system. For interrater reliability, tracers were normed by

comparing their tracings of 10 dendritic systems to the

same tracings completed by the primary investigator (BJ).

Pearson’s product correlations across soma size, TDL,

and DSN (defined below) averaged 0.97, 0.96, and

0.96, respectively. An analysis of variance (ANOVA;

a = 0.05) indicated that there was no significant differ-

ence among tracers in the three measures. Finally, the

primary investigator reexamined all final tracings to

ensure accuracy.

Cell descriptions and dependent dendritic/spine

measures

Deep pyramidal neurons were qualitatively described

according to nomenclature found in previous neuro-

morphological research (e.g., Ngowyang 1932; Ferrer et al.

1986a, b). Descriptions for each neuron used qualitative

criteria, such as cortical location, size, dendritic field

patterns, presence of spines, and soma characteristics.

Nevertheless, because axons were not visible and because

intermediates between different neuronal types exist, these

designations remain tentative.

For quantitative measures, a centrifugal nomenclature

was used (Bok 1959; Uylings et al. 1986): dendritic

branches arising from the soma are first-order segments

until they bifurcate into second-order segments, which

branch into third-order segments, and so on. In addition to

noting soma size (as apparent surface area in lm2) and

soma depth from the pial surface, five previously estab-

lished measures (Jacobs et al. 2001) were used to analyze

each neuron. Total dendritic length (TDL, lm) refers to

the summed length of all dendritic segments. Mean seg-

ment length (MSL, lm) is the average length of each

dendritic segment. Dendritic segment count (DSC) rep-

resents the number of dendritic segments. Dendritic spine

number (DSN) refers to the total number of spines on

dendritic segments. Dendritic spine density (DSD) is the

average number of spines per micron of dendritic length.

Additionally, dendritic diameter throughout the cell was

determined, thus providing a sixth measure: dendritic

volume (Vol, lm3). Although tested independently, many

of these measures are inherently interrelated. Finally,

dendritic branching patterns of all traced neurons were

examined using Sholl analysis (Sholl 1953), which

quantified the number of dendritic intersections at 20-lm

intervals radiating centrifugally from the soma. This

analysis assisted in the characterization of apical and

basilar dendrites.

Independent variables and statistical analyses

For superficial pyramidal neuron comparisons, only basilar

dendrites were examined inferentially because apical den-

drites were often incomplete. Basilar dendritic data were

aggregated by neuron (CELL), and brain region (AREA).

The resulting dataset was analyzed using a one-way anal-

ysis of variance (ANOVA; a = 0.05) to investigate the

effects of area (i.e., frontal and occipital) on each depen-

dent measure for the basilar skirt.

Results

Overview

Nissl-stained sections indicated that the frontal cortex was

slightly thicker than the occipital cortex, with both regions

exhibiting a relatively thick layer III and lacking a distinct

layer IV (Fig. 3; Table 1). The majority of cells traced

resided in layers III and V. In Golgi preparations, the

dominant characteristic across both occipital and frontal

Fig. 3 Photomicrographs of Nissl-stained cortex from frontal (a) and

occipital (b) cortex with labeled layers. Note that the elephant appears

to lack a well-developed layer IV. Scale bar 1 mm

Table 1 Laminar and cortical depth from the pial surface in elephant

cortex (lm)

Frontal region Occipital region

Layer I/II junction 560 ± 14 519 ± 20

Layer II/III junction 730 ± 13 675 ± 19

Layer III/V junction 1,495 ± 14 1,643 ± 38

Layer V/VI junction 2,034 ± 21 2,098 ± 42

Gray/white matter junction 2,828 ± 41 2,737 ± 55

Values represent mean ± SEM

Brain Struct Funct (2011) 215:273–298 277

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regions was the vast heterogeneity of spiny cell types

(Fig. 4). Generally, superficial layers contained many

small, bifurcating variations of the pyramidal cell with

thick apical dendrites that ramified as they ascended toward

the pial surface. Deeper neurons possessed broad basilar

skirts and often extended multiple widely diverging apical

dendrites. In some cells, a prominent basilar dendrite or

taproot penetrated the white matter. Aspiny neurons tended

to possess long dendrites that bifurcated near the soma or

not at all, and were located in upper layer III or lower layer

V. Tracings of individual cell types are provided in Figs. 5,

6, and 7 with quantitative data provided in Table 2. It

should be noted that laminar boundaries are not indicated

in Figs. 5, 6, and 7 because, given the considerable vari-

ability across individual sections, neurons of similar depth

may not necessarily be located in the same cortical layer

across different sections. These descriptions, based on

examination of all Golgi-stained sections as well as the

quantified neurons themselves, are presented below with

superficial pyramidal neurons being considered separately

because they were more extensively analyzed.

Spiny neurons

Deeper pyramidal neurons with ascending apical dendrites

exhibited several variations: magnopyramidal (with and

without a taproot), multiapical, and fork neurons. Magno-

pyramidal neurons (n = 3, Fig. 6F–H), located at a depth

of approximately 1,500 lm in layer III of the occipital

lobe, projected an ascending, typically bifurcating, apical

process, often exceeding 1,200 lm in length. Their somata

varied in size (Table 2) and shape, from small and trian-

gular to very large and fusiform. The basilar skirt extended

in all directions to produce a circular receptive area. The

magnopyramidal cells were generally less spiny than their

superficial counterparts, with a DSD between 0.33 and 0.47

(Table 2). Sholl analysis revealed a high density of basilar

dendrites similar in size and distribution to those of

superficial pyramidal cells, although the apical dendrite

was always longer (Fig. 8a).

An apparent variation of the above was the magnopy-

ramidal-taproot neurons (n = 4, Figs. 4D, 5I–L, 9), which

were located in layers III and V of the frontal lobe. Because

of their prominent appearance in Golgi-stained sections

and their trunk-like taproot, we named these ‘‘matriarch’’

neurons (referring to the matriarch’s prominent role in

elephant society). They exhibited extensive dendritic

branching, which suggests a broad sampling of cortical

information (Brown et al. 2008). The matriarch neuron’s

fusiform soma extended an apical process for up to 900 lm

that often bifurcated during its ascent to the pial surface. A

basilar skirt projected densely and uniformly in all direc-

tions. Unique to this neuron was the addition of a complex,

descending taproot that tended to bifurcate widely into

smaller segments while the main branch extended for long

distances (up to 1 mm) toward the underlying white matter.

Fig. 4 Low magnification photomicrograph of elephant frontal

cortex illustrating a wide diversity of neuronal morphologies: inverted

pyramidal neurons (A, C); superficial pyramidal neurons (B, E);

magnopyramidal-taproot or matriarch neuron (D, also seen in

Fig. 5J); and fork neuron (F, also seen in Fig. 5F). Pial surface is

at the top of the photomicrograph. Scale bar 500 lm

278 Brain Struct Funct (2011) 215:273–298

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DSD ranged from 0.32 to 0.54 (Table 2). In the Sholl

analysis, the matriarch neuron (Fig. 8c) was second only to

the neurogliaform cell (Fig. 7L) in terms of overall den-

dritic density.

Multiapical pyramidal neurons (n = 3, Figs. 5M, 6K–L),

present both in frontal and in occipital cortex, were among

the deepest of the spiny cells, located predominantly in

layer V at a depth of 1,500–1,700 lm (Table 2). Although

often incomplete due to sectioning, multiple, prominent

apical dendrites extended from a spherical cell body to

ascend symmetrically toward the pial surface. Basilar

dendrites extended with some variability from the soma,

either favoring radial processes that descended toward the

white matter or more lateral, tufted projections. Dendrites

possessed a relatively low density of spines, with a DSD

ranging from 0.30 to 0.36 (Table 2). Sholl analysis

revealed two apical dendritic peaks, with the second peak

being somewhat larger (Fig. 8e). Basilar dendritic density

peaked closer to the soma than did the apical dendrite.

A fork neuron (or Gabelzelle; n = 1, Figs. 4F, 5F),

originally described by Ngowyang (1932) in the human

frontoinsular cortex, was found at the boundary between

layers III and V of the frontal cortex. It possessed a split

soma that tapered into two thick, ascending apical den-

drites 600 lm in length. Basilar projections extended

obliquely as fine, parallel dendritic processes. DSD was

relatively high at 0.58 (Table 2). Its axon emerged proxi-

mally from a basilar dendrite and appeared to descend. The

fork neuron was unusual in that, like the horizontal pyram-

idal cell, its apical and basilar branches were similar in

length (Fig. 8g), although the apical dendrites were clearly

truncated by sectioning.

Greater variation in apical dendritic orientation was

observed with horizontal and inverted pyramidal neurons.

Horizontal pyramidal neurons (n = 2, Fig. 6I–J), found in

layer III of the occipital lobe, possessed a narrow, elon-

gated soma from which a quickly ramifying apical dendrite

extended laterally or obliquely (\45�). Basilar dendrites

Fig. 5 Neurolucida tracings of spiny neurons in the elephant frontal

cortex presented to indicate their relative soma depths from the pial

surface (in lm): crab-like neuron (A); superficial pyramidal neurons

(B–E); fork neuron (F); inverted pyramidal neuron (G, H);

magnopyramidal-taproot or matriarch neurons (I–L); multiapical

pyramidal neuron (M). The quantitative dependent measures for all

neurons are provided in Table 2. Scale bar 100 lm

Brain Struct Funct (2011) 215:273–298 279

123

branched into distinct, parallel collaterals or extended

numerous secondary and tertiary dendrites. DSD was

between 0.44 and 0.47 for the two cells (Table 2). In the

Sholl analysis, the apical and basilar dendrites were

roughly equal in density and length (Fig. 8f). Inverted

pyramidal neurons (n = 3, Figs. 4A, C, 5G–H, 6A), found

in layer III of frontal and occipital cortices, possessed

apical dendrites that descended and bifurcated from a

roughly triangular soma. In the frontal lobe, the length of

the apical process varied from 600 to 1,100 lm and pos-

sessed few collaterals. The basilar skirt projected toward

the pial surface, producing a circular receptive area. The

smallest and most superficial of these cells (Fig. 6A) pos-

sessed a short apical shaft that descended obliquely for less

than 400 lm in length, and an axon that emerged from the

base of the soma and bifurcated, one branch ascending and

the other descending. DSD ranged from 0.39 to 0.46

(Table 2). Sholl analyses confirmed the inverted pyramidal

neuron’s (Fig. 8b) structural similarity to the magnopyra-

midal neurons.

Finally, two ‘‘atypical’’ spiny neuron variants appeared

to have horizontally oriented dendritic arbors: flattened

pyramidal and ‘‘crab-like’’ neurons. Flattened pyramidal

neurons (n = 3, Fig. 6M–N, P) were found in deep layer

III and upper layer VI of the occipital cortex, where they

appeared to be fairly common. Flattened pyramidal neu-

rons sent multiple, thick apical dendrites from opposing

ends of the soma to ascend outward and then gradually

toward the pial surface. Somata varied from elongated to

globular. Dendrites extended further laterally than verti-

cally, with basilar dendrites often descending toward the

white matter alongside apical dendrite collaterals. Their

thick dendrites made the cells, by volume, among the

largest in the present sample. Their DSD was similar to

other large pyramidal cells, ranging from 0.37 to 0.41

(Table 2). Sholl analysis revealed the maximum density of

apical dendrites to be further from the soma than that of the

basilar dendrites (Fig. 8d). Crab-like neurons (n = 3,

Figs. 5A, 6B, O) were so designated because their dendritic

branches emerged from opposite ends of a rounded soma to

ramify symmetrically, producing claw-like shapes. These

were located in layers II and III and at the junction between

layer V and VI of the frontal and occipital lobes. DSD

varied greatly, ranging from 0.39 to 0.67 (Table 2). Sholl

analysis indicated that crab-like neurons (Fig. 8j) exhibited

a relatively small dendritic receptive area.

Fig. 6 Neurolucida tracings of spiny neurons in the elephant

occipital cortex presented to indicate their relative soma depths

from the pial surface (in lm): inverted pyramidal neuron (A); crab-like

neurons (B, O); superficial pyramidal neurons (C–E); magnopyramidal

neurons (F–H); horizontal pyramidal neurons (I–J); multiapical pyrami-

dal neurons (K–L); flattened pyramidal neurons (M–N, P). The quanti-

tative dependent measures for all neurons are provided in Table 2. Scalebar 100 lm

280 Brain Struct Funct (2011) 215:273–298

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Aspiny neurons

Aspiny multipolar neurons (n = 6, Figs. 7A–D, J–K) were

found throughout all layers of frontal and occipital cortices.

They varied in appearance, but generally exhibited a small,

globular soma from which a few (2–6) primary, straight den-

dritic projections extended to 1,000 lm in length. In the Sholl

analysis, some multipolar cells (Fig. 8h) exhibited dendritic

projections that were longer than any other cells, excluding the

magnopyramidal and inverted pyramidal neurons.

Bipolar neurons (n = 5, Fig. 7E–I), similar in size and

shape to the multipolar neurons, were found in layers III

and V of frontal cortex. They possessed a small, globular

soma from which two main, thick dendritic segments

extended in opposite directions. Thin collaterals, up to

900 lm in length, extended outward from these segments

and ramified to produce a roughly symmetrical bitufted

appearance. According to the Sholl analysis, the dendrites

of these cells (Fig. 8i) were, on average, shorter and less

dense than the comparable multipolar neurons.

The neurogliaform neuron (n = 1, Fig. 7L), also called a

spiderweb cell by Ramon y Cajal (1922), was an aspiny,

diffusely branching multipolar cell in layers II and III of

occipital cortex. Three main dendritic branches emerged from

an elongated soma and gave rise to numerous, approximately

200 lm dendritic projections that bifurcated densely in all

directions to produce a spherical, net-like receptive area with a

TDL that far surpassed that of any other cell (Table 2). A few

longer dendrites (up to 600 lm in length) extended along the

cell’s vertical axis. This cell had by far the densest dendritic

arbor (DSC = 337), with over twice as many ring intersec-

tions as any other neuron in the Sholl analysis (Fig. 8k).

Quantitatively, three general observations about aspiny

interneurons and spiny neurons in the elephant emerge

(excluding the neurogliaform neuron, which was a clear

dendritic outlier): (1) aspiny neurons in frontal cortex

(MVol = 11,870 ± 5,337 lm3; MTDL = 4,650 ± 1,462 lm)

appeared somewhat larger than those in occipital cortex

(MVol = 7,514 ± 2,441 lm3; MTDL = 2,558 ± 599 lm),

although the small sample size severely limits the value of

Fig. 7 Neurolucida tracings of aspiny neurons found in elephant

frontal (A–I) and occipital (J–L) cortices presented to indicate their

relative soma depths from the pial surface (in lm): multipolar neurons

(A–D, J–K); bipolar neurons (E–I); neurogliaform neuron (L). The

quantitative dependent measures for all neurons are provided in

Table 2. Scale bar 100 lm

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Table 2 Summary statistics for elephant neurons in frontal and occipital cortex

Type Cella Vol.b TDLc MSLc DSCd DSNe DSDf Soma sizeg Soma depthh

Frontal

Crab-like 5A 5,732 4,249 83 51 2,863 0.67 439 424

Superficial pyramidal 5B 8,233 4,574 82 56 2,036 0.45 286 741

Superficial pyramidal 5C 17,880 6,740 95 71 4,162 0.62 478 691

Superficial pyramidal 5D 30,300 7,603 104 98 4,839 0.64 479 818

Superficial pyramidal 5E 17,250 8,233 106 78 5,379 0.65 506 875

Fork 5F 55,256 8,050 117 69 4,685 0.58 1,154 1,436

Inverted pyramidal 5G 13,937 5,634 95 59 2,608 0.46 681 1,101

Inverted pyramidal 5H 18,055 7,370 93 79 3,012 0.41 706 1,203

Matriarch 5I 27,149 7,716 100 77 2,489 0.32 1,061 1,066

Matriarch 5J 33,500 10,838 111 97 5,856 0.54 766 1,299

Matriarch 5K 43,904 8,269 115 72 3,977 0.48 973 1,317

Matriarch 5L 28,360 10,377 113 92 5,430 0.52 799 1,423

Multiapical pyramidal 5M 54,261 6,742 98 69 2,340 0.35 1,552 1,662

Multipolar 7A 10,843 6,879 186 37 – – 387 1,124

Multipolar 7B 9,311 5,152 152 34 – – 611 1,069

Multipolar 7C 11,905 3,778 114 33 – – 801 1,811

Multipolar 7D 24,569 5,839 195 30 – – 1,375 2,027

Bipolar 7E 13,497 5,883 115 51 – – 757 1,811

Bipolar 7F 9,186 4,129 118 35 193 0.05 494 1,132

Bipolar 7G 8,510 3,996 125 32 – – 351 1,203

Bipolar 7H 5,851 1,946 97 20 – – 411 1,893

Bipolar 7I 13,160 4,248 112 38 – – 737 1,641

Occipital

Inverted pyramidal 6A 5,173 3,465 96 36 1,346 0.39 398 823

Crab-like 6B 4,596 2,500 104 24 1,214 0.49 407 806

Superficial pyramidal 6C 10,897 5,921 82 72 3,346 0.57 505 722

Superficial pyramidal 6D 16,264 5,509 115 48 3,630 0.66 643 746

Superficial pyramidal 6E 11,312 6,752 83 81 3,914 0.58 256 650

Magnopyramidal 6F 18,924 8,752 115 76 4,094 0.47 780 1,470

Magnopyramidal 6G 76,591 7,925 113 70 3,148 0.40 2,015 1,487

Magnopyramidal 6H 46,090 6,895 90 77 2,296 0.33 2,004 1,525

Horizontal pyramidal 6I 29,131 7,554 113 67 3,521 0.47 988 1,571

Horizontal pyramidal 6J 28,811 6,411 88 73 2,853 0.44 941 1,090

Multiapical pyramidal 6K 35,055 4,428 84 53 1,311 0.30 1,562 1,536

Multiapical pyramidal 6L 32,114 6,375 133 48 2,291 0.36 1,284 1,734

Flattened pyramidal 6M 58,109 6,187 115 54 2,293 0.37 1,694 1,456

Flattened pyramidal 6N 36,137 4,346 92 47 1,706 0.39 1,222 1,407

Crab-like 6O 13,679 4,938 60 83 1,949 0.39 689 2,003

Flattened pyramidal 6P 57,494 6,476 93 70 2,674 0.41 1,092 2,233

Multipolar 7J 9,241 2,981 110 27 210 0.07 479 672

Multipolar 7K 5,788 2,134 71 30 34 0.02 423 704

Neurogliaform 7L 26,959 14,818 44 337 – – 857 528

a Refers to tracings of individual cells as indentified in Figs. 5, 6, 7b Volume in lm3

c Length in lmd Number of segments per neurone Number of spines per neuronf Number of spines per lm of dendritic lengthg Soma size in lm2

h Soma depth in lm from the pial surface

282 Brain Struct Funct (2011) 215:273–298

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this comparison; (2) the aspiny interneurons (MVol =

11,078 ± 5,147 lm3; MTDL = 4,270 ± 1,569 lm) exhib-

ited substantially less overall dendritic extent than the

spiny neurons (MVol = 28,765 ± 11,078 lm3; MTDL =

6,580 ± 1,906 lm); and (3) the aspiny interneurons

exhibited considerably longer (MMSL = 127 ± 37 lm),

but fewer (MDSC = 33 ± 8) dendritic branches than did the

spiny neurons (MMSL = 100 ± 15 lm; MDSC = 67 ± 17).

Superficial pyramidal neurons

Superficial pyramidal neurons (n = 40; Figs. 4B, E, 5B–E,

6C–E) were well impregnated across both occipital and

frontal cortices (Fig. 10), facilitating a more detailed

quantitative analysis. These neurons were sampled pri-

marily from upper cortical layer III with minimal variation

of soma depth across cortical areas (frontal 818 ± 108 lm;

occipital 769 ± 149 lm). Measurements of soma size

indicated minimal variation between cortical areas (frontal

515 ± 80 lm2; occipital 517 ± 156 lm2). There was a

significant positive correlation between soma depth and

soma size (r(40) = 0.451, p \ 0.01), as well as between

soma size and volume (r(40) = 0.353, p \ 0.05), which

underscored the importance of maintaining similar soma

depths between areas for subsequent comparisons.

In terms of appearance, superficial pyramidal neurons

exhibited typical basilar skirts, which projected radially

for a short distance from the soma. They were charac-

terized by apical dendrites that generally bifurcated

around 50 lm from the soma to form two, ascending

apical shafts (Figs. 5D–E, 6C, E, 10a, d), or by two

diverging apical dendrites (Figs. 5C, 6D) that emerged

directly from the soma. These resulting apical trunks

traveled obliquely to the pial surface, thus creating a large

apical arbor for each cell and a crisscross or ‘‘V’’

appearance in the upper layers (Fig. 11b–c). Generally,

these branches appeared to bundle with other branches

traveling at similar angles toward the pial surface

(Fig. 11a, d).

Dendritic morphology-dependent measures

Selected dependent measures for basilar data were first

analyzed using a MANOVA test statistic. Due to direct

Fig. 8 Sholl analyses of 11 cell types assessing the relative

complexity of basilar, apical, and total dendritic branching patterns.

Concentric rings, separated by 20 lm increments and centered on the

soma, were used to measure dendritic intersections. Neurons a–g were

spiny. Neurons h–k were aspiny. a–c exhibited relatively long apical

dendrites, d–g had shorter apical dendrites. For cells with basilar

dendrites, the density of basilar intersections peaked before 200 lm,

as was the case for the total dendritic density of all aspiny neurons.

The apical dendrite either tended to extend for a much larger distance

than the basilar dendrites (a, b) or was nearly equal to them (c–g).

Aspiny neurons (h–j) generally had low dendritic densities, the

notable exception being the neurogliaform neuron (k), which had the

highest dendritic density of all neurons. Asterisks represents that the

y-axis for the neurogliaform graph (k) is set to a larger scale than

other cells to compensate for its particularly high dendritic density

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associations between DSD and DSC with other measures,

these variables were not used in the MANOVA. The

MANOVA was significant (F(4, 35) = 3.125, p \ 0.05,

gp2 = 0.263). Post hoc analyses, consisting of six one-way

ANOVAs, were then conducted for each dependent vari-

able. All dependent measures, except MSL and DSD, were

significantly higher in frontal cortex, indicating the pres-

ence of more complex basilar dendrites. Although there

were not enough complete apical branches to warrant sta-

tistical tests, descriptive data indicated a similar pattern for

apical dendrites. The differences in complexity can be seen

visually in Fig. 12, which contains representative tracings

from each region.

Volume

Basilar dendritic volume was significantly greater (F(1, 38)

= 5.80, p \ 0.05, gp2 = 0.132) in frontal than in occipital

cortex (by 25.2%; Fig. 13a). On average, neurons from

frontal cortex contained 29.5% thicker apical dendrites and

23.3% more overall volume than neurons from occipital

cortex.

Dendritic length

Basilar dendrites in frontal cortex exhibited significantly

greater TDL (F(1, 38) = 7.15, p \ 0.05, gp2 = 0.158) than

basilar dendrites in occipital cortex (Fig. 13b). The trend

was in the opposite direction for MSL, but was not sig-

nificant (Fig. 13c). Both TDL (by 19.0%) and MSL (by

5.0%) were greater for apical dendrites in the frontal cortex

than in the occipital cortex. Descriptive data from complete

neurons (i.e., basilar and apical dendrites combined)

showed that, overall, TDL was greater (by 13.5%) and

MSL was shorter (by 9.2%) in the frontal cortex than in the

occipital cortex.

Dendritic segment count

There was a significant difference in basilar DSC across

both areas (F(1, 38) = 10.93, p \ 0.05, gp2 = 0.223), with

basilar dendrites in frontal cortex exhibiting 25.1% higher

DSC than occipital cortex (Fig. 13d). Apical dendrites also

appeared to have higher DSC in frontal cortex (by 14.8%),

and complete neurons in frontal cortex had 20.7% higher

DSC than those in occipital cortex.

Dendritic spine number and density

Basilar dendrites of neurons in the frontal cortex displayed

a significantly higher DSN (by 25.1%; Fig. 13e) than

neurons in the occipital cortex (F(1, 38) = 7.95, p \ 0.01,

gp2 = 0.173). DSD was not significant (Fig. 13f). Apical

dendrites in frontal cortex contained 29.4% more spines

and had a 13.4% higher DSD than those in occipital cortex.

Evidence from complete neurons indicated that frontal

Fig. 9 Photomicrographs of two magnopyramidal-taproot or matri-

arch neurons (a, b) with higher magnification insets of their spine-rich

ascending apical (a) and descending taproot (t) dendrites. Neurolucida

tracings can be seen in Fig. 5L for a, and in Fig. 5I for b. Scale bar100 lm for a and b, and 25 lm for the insets

284 Brain Struct Funct (2011) 215:273–298

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Fig. 10 Photomicrographs of Golgi-impregnated superficial pyrami-

dal neurons in occipital (a) and frontal (d) cortices. Associated

photomicrographs of apical (occipital: b; frontal f) and basilar

(occipital: c; frontal e) dendrites are also shown at higher

magnifications. Note in both cortical regions the presence of

bifurcating apical dendrites. For a, b, d, f scale bars 50 lm. For c,

e scale bars 25 lm

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superficial neurons contained 20.2% more spines and had a

7.6% higher DSD than did occipital neurons.

Sholl analysis

Frontal and occipital regions had similar shapes in Sholl

analysis profiles, but frontal results were slightly

higher on average (Fig. 14a, b). Basilar dendrites in both

areas reached their maximum complexity (frontal

16.45 ± 0.99 intersections; occipital 12.80 ± 0.86 inter-

sections) 120 lm from the soma, and apical dendritic

complexity peaked around 200 lm from the soma

(frontal 4.88 ± 0.50 intersections; occipital 4.10 ± 0.48

intersections).

Comparison to human supragranular pyramidal neurons

Elephant superficial pyramidal neurons were compared to

supragranular pyramidal neuron data gathered with similar

methodology in human inferior frontal (specifically, area

11, from Jacobs et al. 2001) and occipital regions (area 18,

from Jacobs et al. 1997). These regions were chosen to be a

relatively close topographic match for those examined in

the elephant. The human supragranular pyramidal neurons

Fig. 11 Photomicrographs of V-apical bundling within elephant

occipital cortex. At lower magnifications (b and c), a V-shaped

crossing pattern resulting from bifurcating apical dendrites is

apparent. At higher magnifications (a and d), apical dendrites appear

to bundle together as they obliquely approach the pial surface. For

a and b scale bars 50 lm. For c scale bar 100 lm. For d scale bar25 lm

286 Brain Struct Funct (2011) 215:273–298

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(n = 116) were traced in 6 neurologically normal subjects

(4 men, 23–69 years of age; 2 women, 32 and 34 years of

age).

Dendritic length and segment count

On average, elephant superficial pyramidal neurons dem-

onstrated only slightly higher TDL than human neurons

both in frontal (by 6.8%) and in occipital (by 2.9%) cor-

tices (Fig. 15a). However, TDL was manifested differently

in humans and elephants, as revealed by MSL and DSC

values. Basilar dendrites in the elephant exhibited greater

MSL both in frontal (by 23.3%) and in occipital cortices

(by 35.6%; Fig. 15b) than they did in humans. In contrast,

elephant DSC was considerably lower than human DSC

(by 16.0% in frontal cortex, and by 33.5% in occipital

cortex; Fig. 15c).

Dendritic spines

Elephant superficial pyramidal neurons exhibited greater

values for DSN and DSD than did human neurons. In

elephant frontal cortex, basilar dendrites exhibited 63.6%

higher DSN and 61.7% higher DSD than in humans. In

elephant occipital cortex, basilar dendrites exhibited 52.3%

higher DSN and 49.9% higher DSD than in humans.

Sholl analysis

Although Sholl analysis profiles were similar across both

area 11 and area 18 in human, they varied considerably

from those in the elephants (Fig. 14). On average, elephant

basilar dendrites extended 760 lm from the soma in frontal

cortex and 740 lm from the soma in occipital cortex. In

contrast, human basilar dendrites extended 380 lm from

the soma in the frontal cortex and 400 lm in occipital

cortex. At the high point of dendritic intersections, human

supragranular pyramidal neurons contained more intersec-

tions than did elephant superficial pyramidal neurons. In

frontal cortex, human basilar dendrites peaked at 22.90 ±

0.90 intersections, whereas elephant dendrites peaked at

16.45 ± 0.99 intersections. In occipital cortex, human

basilar dendrites peaked at 21.47 ± 0.65 intersections,

whereas elephant dendrites peaked at 12.80 ± 0.86 inter-

sections. Overall, the Sholl analysis profiles confirm that

human supragranular pyramidal neurons were more con-

densed and contained more branch points than the elephant

superficial pyramidal neurons.

Fig. 12 Sample tracings of superficial pyramidal neurons, and their

corresponding dendritic measurements, from frontal (cells a–d) and

occipital (cells e–h) cortices. Neurons are presented to indicate their

relative soma depths from the pial surface (in lm). Within each

cortical area, the two neurons with relatively complete apical

dendrites appear on the left. Note also the bifurcation of all apical

dendrites. In general, frontal neurons appeared more dendritically

complex than occipital neurons. Scale bars 100 lm

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Discussion

The present study is the first to document neuronal

morphology in the African elephant cortex. In terms of

cytoarchitecture, the elephant neocortex is characterized

by the presence of only five cortical layers as it altogether

lacks a visible layer IV, which is quite different from

what is observed in primates. Although it has not been

studied in much detail to date, especially in terms of

regional differences, we can make several generalizations.

Layer I appears to be relatively thick and more cellular

than in most terrestrial species. Layer II is clearly visible,

densely packed, and displays cellular clustering in many

cortical regions, particularly in the insula (Hakeem et al.

2009), as has been reported in cetaceans (Manger et al.

1998; Hof and Van der Gucht 2007). Layer II also con-

tains occasional large pyramid-like neurons. Layer III is

relatively thick and populated by large pyramidal neurons,

the density and size of which clearly vary among different

cortical domains; this fact deserves a detailed investiga-

tion. Layer V presents as a relatively thin row of very

large pyramidal cells usually distributed in small groups,

presumably representing principal efferent neurons, and a

thicker deep portion containing smaller pyramidal cells.

Layer VI is of variable thickness among cortical regions

and contains a variety of pyramidal and non-pyramidal

neurons as seen in many other mammalian species. This

cortical lamination pattern with lack of an internal gran-

ular layer IV may reflect a particular organization of

cortical connectivity in elephants, perhaps comparable to

that in cetaceans, where agranularity is also a neocortical

characteristic (Hof et al. 2005; Hof and Van der Gucht

2007). Although, we are currently exploring the cytoar-

chitecture of elephant cortex in detail, it remains clear

that complex neocortical gyrification and distinct laminar

organization represent remarkable features of the elephant

brain.

In terms of neuromorphology, elephant cortex featured a

great diversity of large, complex neurons, with consider-

able variety exhibited by ‘‘atypical’’ spiny neurons. A

prominent characteristic in elephant cortex was the

V-shaped arrangement of bifurcating apical dendrites.

Quantitatively, the dendrites of superficial pyramidal neu-

rons in the elephant frontal cortex were more complex than

those in occipital cortex. In comparison to humans, ele-

phant superficial pyramidal neurons exhibited similar

overall basilar dendritic length, but individual dendrites

tended to be longer in the elephant with less intricate

branching. Before considering the implications of these

findings, methodological issues must be addressed.

Fig. 13 Bar graphs of relative volume (a), total dendritic length (b),

mean segment length (c), dendritic segment count (d), dendritic spine

number (e), and dendritic spine density (f) of superficial pyramidal

neuron basilar dendrites in elephant frontal and occipital cortex. The

basilar dendrites in frontal cortex had significantly greater volume,

TDL, DSC, DSC, and DSN than did those in occipital cortex. Errorbars represent SEM

288 Brain Struct Funct (2011) 215:273–298

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Methodological considerations

In general, the same constraints that apply to Golgi-stained

human tissue also apply here (Jacobs and Scheibel 2002):

(1) a small sample size in terms of subjects and sampled

neurons (Jacobs and Scheibel 1993); (2) lack of historical

information on the subjects (Jacobs et al. 1993); (3)

underestimation of spines in light microscopy (Horner and

Arbuthnott 1991); and (4) inherent issues with Golgi

impregnations (Braak and Braak 1985). These general

limitations are accepted and we focus here on three broader

issues.

Functional classification of elephant cortex and regional

specialization

Functional characteristics of the sampled regions could not

be determined. The frontal cortex, although located in what

appear to be anterior orbital gyri, could subserve prefrontal

executive functions (Barbas 1995) or, equally likely,

premotor activities. The function of the occipital cortex is

even more problematic, as this region could support visual

or even auditory processes. As such, it is impossible to

correlate dendritic/spine measure with the functional

attributes of these regions, as has been done in primates

(Elston and Rosa 1998a, b; Jacobs et al. 2001). Moreover,

it is not possible to make definitive statements about the

regional distribution of traced neurons in the present study

despite observing that some neuron types appeared only in

one cortical region.

Neuromorphological nomenclature

Unambiguously classifying neurons is problematic when

the criteria are not clearly defined (Bota and Swanson

2007), are too restrictive (Germroth et al. 1989), or when

cellular morphologies form a continuum rather than distinct

categories, as may be the case for extraverted telencephalic

neurons (Sanides and Sanides 1972; Ferrer and Perera

1988). Further classification issues emerge when definitions

Fig. 14 Graphic representation of Sholl analysis results for elephant

superficial and human supragranular pyramidal neurons, showing

mean numbers of dendritic intersections in elephant frontal (a),

elephant occipital (b), human frontal (c), and human occipital

(d) cortices. Although dendritic extent between areas and species is

similar, elephant dendrites are distributed across a greater distance

from the soma and exhibit fewer intersections at the peak than

observed in humans. Error bars represent SEM

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change over time, as typified by the term ‘‘pyramidal,’’

which now applies to a much broader category of neurons

than it did originally (Masland 2004). Compounding the

classification problem is an essentially Euarchontoglires-

centric nomenclature in the literature, whereby the

(anthropoid) primate together with the (murid) rodent brain

constitutes the template against which all other brains are

compared, thus dictating what is ‘‘typical’’ versus ‘‘atypi-

cal’’ (Manger et al. 2008). In the present study, we have

attempted to follow existing nomenclature where possible

or, when appropriate, named cells based loosely on their

dendritic morphology (e.g., crab-like neuron). Although we

observed many examples of the presented neurons in our

examination of the Golgi-stained sections, we only traced

those that were relatively complete and unobscured, which

limits our overall conclusions regarding neuronal types.

Finally, the present study was also limited because it

described neurons based only on somatodendritic measures.

Comparison of human and elephant dendritic/spine

measures

With regard to dendritic measures, only portions captured

in the 120-lm thick section could be compared. Because

the basilar dendrites in elephants exhibited greater TDL

than in humans, the observed TDL difference between the

two species was probably attenuated (Jacobs et al. 1997).

Nevertheless, sampling within an equal volumetric ‘‘slice’’

provides useful relative measures for comparison. With

regard to spine measures, several issues prohibit a

Fig. 15 Bar graphs displaying the differences in relative total

dendritic length (a), mean segment length (b), and dendritic segment

count (c) for basilar dendrites in elephant superficial pyramidal

neurons and human supragranular pyramidal neurons across frontal

and occipital cortex. Sample tracings of elephant superficial and

human supragranular pyramidal neurons are also provided. Although

elephant and human pyramidal neurons show similar TDL values,

human neurons contain shorter segments and more intricate branch-

ing. All human data were obtained from Jacobs et al. (1997) for

occipital cortex (specifically area 18), and Jacobs et al. (2001) for

frontal cortex (specifically area 11). Error bars represent SEM

290 Brain Struct Funct (2011) 215:273–298

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meaningful interpretation of the relative DSN and DSD

values. First, the human brains were immersion-fixed,

whereas the elephant brains were perfusion-fixed, the latter

revealing greater detail (i.e., spines; Morest and Morest

2005). Second, autolysis time in the humans was longer

(*14 h) than in the elephants (*2 h), which potentially

decreased the number of impregnated spines in the human

samples (de Ruiter 1983). Finally, although data were

collected in a methodologically similar manner, human

neurons were traced using a Neurolucida Lucivid system,

whereas elephant neurons were traced on a Neurolucida

camera system. It was recently discovered that the higher

magnification in the camera system increases the number

of spines identified by about 17% (Anderson et al. 2010),

clearly making accurate spine comparisons between the

two species problematic.

Spiny neurons

The elephant cortex is characterized by an even greater

variety of spiny neurons than the morphologically hetero-

geneous inferior temporal cortex of macaque monkeys and

rats (Germroth et al. 1989; De Lima et al. 1990). Although

several of these neuron types have been observed in other

eutherian species, what remains noteworthy in the elephant

is their arrangement, which represents a striking departure

from the vertical apical dendrites and cortical columns that

have been deemed the fundamental, if not canonical

building block of the cerebral cortex (Mountcastle 1997;

Innocenti and Vercelli 2010).

These spiny cortical neurons exist along a continuum

from those that appear more pyramid-like to those that

radically differ from the ‘‘typical’’ pyramidal neuron in

terms of morphology and/or orientation. In the elephant,

three neuron types, in addition to superficial pyramidal

neurons, approximate general pyramidal neuron morphol-

ogy: magnopyramidal, multiapical, and fork neurons. The

only exact comparison point in the literature is the single

Indian elephant neuron depicted in Barasa and Shochato-

vitz (1961), which is consistent with the present layer V

magnopyramidal neurons. Although pyramidal neurons in

the cat, dog, pig, and sheep resemble those in the elephant,

the most striking similarities are found in the cow, horse,

two-toed sloth, and anteaters (Barasa 1960; Ferrer et al.

1986b; Sherwood et al. 2009), which exhibit prominent,

bifurcating apical dendrites much the same as those

observed in elephant magnopyramidal and multiapical

neurons. Unfortunately, size comparisons with cow and

horse neurons are not possible because Barasa (1960) did

not provide scale bars. The fork neuron has been observed

in the insula and hippocampus of humans, chimpanzees,

and orangutans (Ngowyang 1936; de Crinis 1934).

Although originally considered a unique type of neuron,

subsequent investigations in several mammals (e.g., pri-

mates, carnivores, ungulates, and rodents) have concluded

that it is a type of enveloping neuron (or Umfassungszelle)

and thus is merely a variant of pyramidal neurons (Juba

1934; Syring 1956). Finally, elephant magnopyramidal,

multiapical, and fork neurons appear at least as dendritic-

ally complex as infragranular pyramidal neurons in the cat

parietal cortex (Yamamoto et al. 1987), human cingulate,

and motor cortices (Meyer 1987; Schlaug et al. 1993).

Within a more polymorphic subgroup of pyramidal

neurons are the horizontal and inverted pyramidal neurons

(Ramon y Cajal 1891; Van der Loos 1965). In terms of

morphology, these are similar to magnopyramidal and

multiapical neurons, but are slightly smaller in dendritic

extent and differ in their specific orientations. Although

horizontal pyramidal neurons have typically been noted in

superficial and deep cortical layers in primates and rodents

(Meyer 1987; Miller 1988), elephant horizontal pyramidal

neurons were located in layer III. These strongly resembled

one variant of the asymmetrical pyramidal neurons in

inferior temporal cortex (Fig. 8e of De Lima et al. 1990),

although one could argue that what De Lima et al. refer to

as a prominent, laterally oriented, basilar dendrite is actu-

ally the apical dendrite. The elephant variant appears to be

a more dendritically complex version than what has been

noted in layer VI of both lissencephalic and gyrencephalic

brains (Ferrer et al. 1986a, b, 1987). Although the function

of these neurons remains unclear, it is possible that they

contribute to the lateral integration of synaptic input (Van

Brederode et al. 2000), which has been suggested as a

major step in neocortical evolution (Ferrer et al. 1986b).

Much the same as horizontal pyramidal neurons, elephant

inverted pyramidal neurons were located in layer III. In

human temporal cortex, they have been documented in

layer V (Ong and Garey 1990). In chimpanzees, inverted

pyramidal neurons are located in layers III, V and espe-

cially VI of sensorimotor cortices, constituting a small

percentage (\1%) of all pyramidal neurons (Qi et al. 1999).

In rats, rabbits, cats, sheep, sloths, anteaters, rock hyrax,

and elephant shrews, they also tend to be located in in-

fragranular layers and constitute a small (1–8.5%) per-

centage of all neurons in the cortex (Parnavelas et al. 1977;

Mendizabal-Zubiaga et al. 2007; Sherwood et al. 2009).

These excitatory neurons have been shown to project to

ipsi- and contra-lateral cortical regions, as well as to the

claustrum and striatum (Bueno-Lopez et al. 1991; Mendi-

zabal-Zubiaga et al. 2007). In the elephant, however, the

extent to which inverted pyramidal neurons share connec-

tional and/or functional attributes with their counterparts in

other species remains unclear.

One type of elephant neuron that does not have a clear

counterpart in the literature is the magnopyramidal-taproot

or ‘‘matriarch’’ neuron of the frontal cortex. Although they

Brain Struct Funct (2011) 215:273–298 291

123

resemble, because of the descending taproot, some of the

layer VI vertical fusiform projection and asymmetric

pyramidal neurons described in macaque inferior temporal

gyrus (De Lima et al. 1990) and some of the layer VI

pyramidal neurons in human motor cortex (Meyer 1987),

they exhibited a much more extensive dendritic system.

Indeed, these were among the most complex neurons in the

present sample, approaching the dendritic extent of human

Betz or Meynert neurons. Although morphologically very

idiosyncratic, some Betz cells, like matriarch neurons,

possess a bifurcating apical shaft with branches that spread

laterally (Braak and Braak 1976), and may also exhibit a

long, descending taproot dendrite (Scheibel and Scheibel

1978a). However, unlike Betz cells, matriarch neurons

were consistently very spiny along both thin and thick

dendrites, including the taproot. Although the function of

matriarch neurons remains unknown, they may represent

extreme adaptations of pyramidal neurons, much the same

as other large cortical neurons in primates (e.g., Betz,

Meynert, and von Economo’s spindle cells; Nimchinsky

et al. 1995; Sherwood et al. 2003; Wittenberg and Wang

2007). Their morphology and potential regional localiza-

tion in the frontal cortex suggests that, like Betz cells,

matriarch neurons may sample a wide range of cortical

input and exert modulatory field effects in the surrounding

neuropil, potentially contributing to associative cognitive

processes (Kaiserman-Abramof and Peters 1972; Scheibel

and Scheibel 1978a).

Among the more ‘‘atypical’’ spiny neurons in elephant

cortex were the horizontally oriented crab-like neurons and

the flattened pyramidal neurons. One could also describe

the three crab-like neurons as horizontally oriented bitufted

neurons; similarly, one cannot rule out that the deeper of

these, in layer VI of occipital cortex (Fig. 6O), may even

have been an unusual, apical-less variant of the solitary cell

of Meynert or of the Meynert–Cajal neuron (von Economo

and Koskinas 1925). Both the crab-like and flattened

pyramidal neurons appear to be much larger variants of

neurons described in the dog and sheep as pyramidal

neurons with multiple horizontal collaterals or horizontal

pyramidal neurons (Figs. 6 and 9 of Ferrer et al. 1986b).

The flattened pyramidal neuron is also strongly reminiscent

of the large, widely bifurcating neuron observed in the cow

(Fig. 7 in Barasa 1960), but with a more pronounced lateral

extension of the apical dendrites (up to 1 mm, Fig. 6M).

The horizontal nature of these two neuronal types may

argue against a strict vertical, columnar arrangement of

cortex (Lubke et al. 2000, 2003), and again suggests a role

for lateral integration of information, perhaps related to the

low density of elephant cortical neurons (Hart et al. 2008).

What remains unclear, however, is why flattened pyramidal

neurons appeared to be more prominent in the occipital

cortex.

Aspiny neurons

Classification of aspiny interneurons typically incorporates

axonal morphology (Lund and Lewis 1993; Petilla Inter-

neuron Nomenclature Group 2008; Druga 2009), which

can be further supplemented with electrophysiological and

molecular properties (DeFelipe 1997; Zeitsev et al. 2009).

Aspiny interneurons in the present study, however, could

only be described according to their general somatoden-

dritic characteristics because axonal arbors were not

revealed by the Golgi stain. Despite this limitation, it

appeared that frontal aspiny neurons exhibited greater

dendritic spread than did those in occipital cortex. Apart

from this size difference, all elephant aspiny interneurons

could be categorized as either multipolar, bipolar, or neu-

rogliaform neurons. With the exception of one aspiny

interneuron (Fig. 7G), which had vertically oriented den-

dritic branches, all other aspiny interneurons contained a

sparse number of laterally or radially extending dendrites.

Dendritic spread in elephant aspiny interneurons, at least

in frontal cortex (with a radius of up to 1,000 lm from the

soma) was extensive. Comparatively, dendritic radii for

local circuit neurons have been reported to be approxi-

mately 50–400 lm in the rat (Kawaguchi 1995) and the cat

(Peters and Regidor 1981; Somogyi et al. 1983),

100–300 lm in the striped dolphin (Stenella coeruleoalba,

Ferrer and Perera 1988), 100–400 lm in the echidna

(Hassiotis and Ashwell 2003), and up to 500 lm in

macaque monkey (Lund and Lewis 1993) and humans

(Kisvarday et al. 1990), although aspiny interneurons in

human motor cortex have been reported with radii of up to

800 lm (Meyer 1987). Similarly, the elephant’s neuro-

gliaform neuron was morphologically similar to what has

been reported in other species, but was approximately 2–3

times larger in terms of its dendritic field (Jones 1984;

Povysheva et al. 2007). Thus, although aspiny interneuron

morphology appears to be highly conserved among

eutherian mammals (Ferrer 1986; Sherwood et al. 2009)

and in the monotreme echidna (Hassiotis et al. 2005), the

elephant variants, at least in the frontal lobe, appear to be

relatively large in terms of dendritic extent. This large size

may be required for these interneurons to exert the wide-

spread inhibitory influences required to shape the temporal

flow of information during cortical processing (Constan-

tinidis et al. 2002).

Elephant superficial pyramidal neurons and human

supragranular pyramidal neurons

Among the most spiny of cortical neurons in the elephant,

superficial pyramidal neurons were ‘‘atypical’’ insofar as

they possessed an apical dendrite with a primary bifurca-

tion at or near the soma, resulting in two obliquely

292 Brain Struct Funct (2011) 215:273–298

123

ascending secondary branches. In the literature, there

appears to be little agreement on whether these bifurcating

apical branches are characteristic of bitufted (Bartesaghi

et al. 2003), multiapical (Ferrer et al. 1986a, b), or even

extraverted pyramidal neurons (Fitzpatrick and Henson

1994). Although resembling layer II extraverted pyramidal

neurons in several other species (hedgehog, bat, and

opossum: Sanides and Sanides 1972; dolphin: Glezer and

Morgane 1990; quokka: Tyler et al. 1998; humpback

whale: Hof and Van der Gucht 2007; giant elephant shrew,

giant and lesser anteater: Sherwood et al. 2009), elephant

superficial pyramidal neurons did not meet the strict defi-

nition of extraversion, whereby apical dendritic extent

exceeds that of basilar dendrites (Sanides and Sanides

1972). In general, the elephant superficial pyramidal neu-

rons possessed a very well-developed basilar skirt, the TDL

of which exceeded that of the apical dendrite in 35 of the

40 traced neurons. Because the basilar array is considered

to be the most progressive feature of the pyramidal neuron

(Sanides and Sanides 1972), it is possible that elephant

superficial pyramidal neurons represent an intermediary

between extraverted neurons and ‘‘typical’’ primate pyramidal

neurons. This could be an adaptation that accompanies a large

brain as an extensive basilar skirt could compensate for the

relatively low neuron density in elephant cerebral cortex

(Tower 1954) by increasing the neuron’s receptive surface

area.

Similarly, the bifurcating apical dendrite greatly

increases the lateral spread of apical branches and could

represent an adaptation to the cytoarchitecture of the ele-

phant neocortex which, as in the hedgehog (Valverde and

Facal-Valverde 1986), bat (Ferrer 1987), and cetaceans

(Garey et al. 1985), does not contain a developed layer IV.

Such laminar organization is associated with an increase in

thalamic inputs into the molecular layer over the deeper

layers (Ferrer and Perera 1988; Glezer and Morgane 1990).

Bifurcating apical dendrites formed V-shaped apical bun-

dles, which is consistent with the description of small-

medium pyramidal cells in cows and horses (Barasa 1960),

but differs substantially from the canonical vertical apical

bundle-type architecture that typifies primate and rodent

neocortex (Fleischhauer et al. 1972). The resulting apical

dendritic bundling, variations of which have been observed

in other species (cat: Fleischhauer 1974; rat: Wyss et al.

1990; tenrec, red-eared pond turtle: Schmolke and Kunzle

1997; echidna: Hassiotis et al. 2003, 2005), raises inter-

esting questions regarding neocortical computation in

cognitively sophisticated species such as the elephant.

Although dendritic bundles appear to help synchronize

signals involved in cortical processing (Scheibel and

Scheibel 1975; Roney et al. 1979; Geisler et al. 2000), their

exact functions remain elusive. It is possible that this V-

shaped arrangement represents a variation of the vertical

column or serves to facilitate communication across wider

domains of cortex, either independent of columns or

between columns that are more widely spaced than in other

species, as has been noted in whales (Hof and Van der

Gucht 2007).

The regional differences observed in elephant superficial

pyramidal neurons, with frontal neurons exhibiting more

complex dendritic systems than occipital neurons, are

generally consistent with findings in human supragranular

pyramidal neurons, where basilar dendrites in area 10 were

found to be more complex than those in area 18 (Jacobs

et al. 1997), and where greater dendritic/spine complexity

has been documented in heteromodal (areas 6b, 39) and

supramodal (areas 10, 11) cortices than in primary (areas

3-1-2, 4) and unimodal (areas 22, 44) cortices (Jacobs et al.

2001). Nevertheless, further research is required in the

elephant to determine if there are progressive increases in

basilar dendritic complexity along hierarchically arranged

pathways, as has been demonstrated in non-human primates

(Elston et al. 1996; Elston and Rosa 1997, 1998a, b), or if

regional differences in basilar dendritic extent are due to

intrinsic properties within each cortical region, as appears to

be the case in the mouse (Benavides-Piccione et al. 2006).

Finally, although TDL was similar between elephants

and humans, there was a difference in how this overall

length was distributed within the neuron. Human supra-

granular pyramidal neurons appeared smaller and denser,

with more complex branching patterns, whereas elephant

superficial pyramidal neurons appeared larger and more

expansive in terms of dendritic spread. Thus, although the

elephant brain is *3 times larger than the human brain,

elephant cortical neurons are not simply ‘‘scaled up’’ ver-

sions of a general mammalian type. As such, simple

models of neuron scaling and computations across phy-

logeny may be problematic (Wittenberg and Wang 2007).

Each species has its own phylogenetic heritage and func-

tional design principles (Harrison et al. 2002). Elephant

neuromorphology may reflect a particular phylogenetic

heritage and/or may be associated with the lower cell

packing density in the cortex. Indeed, the elephant cortex

appears to have a lower neuronal density than dolphins and

primates (Haug 1987; Lyck et al. 2009). These data,

combined with calculations of total volume of cortex

dedicated to somatic processing (Hofman 1982), suggest

that elephants have approximately 10 billion non-somatic

neurons spread across a large area of neocortex, compared

to 6.5 billion tightly packed neurons for chimpanzees and

20 billion tightly packed neurons for humans (Hart et al.

2008). As such, it has been suggested that elephant neo-

cortex may favor long-distance connections over the more

compartmentalized primate model, a possibility tentatively

supported by the longer basilar dendritic systems docu-

mented here.

Brain Struct Funct (2011) 215:273–298 293

123

Evolutionary and cognitive considerations

The present results confirm that the African elephant

cerebral cortex contains a rich variety of large neurons with

expansive, spine-rich dendritic systems and, presumably,

long axonal projections to interconnect these relatively

sparsely packed neurons (Wang et al. 2008). These findings

reinforce the high metabolic expense of large brains

(Grande 1980), and appear consistent with evidence in the

elephant neocortex for high glia-neuron ratios (Haug

1987), high neocortical white–gray matter ratios (Zhang

and Sejnowski 2000), and evolutionary selection on aero-

bic energy metabolism genes (Goodman et al. 2009).

Reasons for the extensive dendritic spread of elephant

neurons, as opposed to the more compact, branched pri-

mate model, remain to be explained.

Perhaps, the most distinguishing characteristic of the

elephant cortex is the presence of widely bifurcating apical

dendrites that branch close to the soma. This seems con-

sistent with the closest living relatives of elephants,

including other afrotherians and especially xenarthrans

(e.g., the giant anteater), but contrasts with findings in the

rock hyrax, where pyramidal neurons have single apical

dendrites resembling those in rodents and primates (Sher-

wood et al. 2009; Bianchi et al, in review). Variations of

bifurcating apical dendrites also appear in cetaceans (Ferrer

and Perera 1988; Hof et al. 1992). More generally, bifur-

cating apical branches appear in caniforms and perisso-

dactyls (Barasa 1960), which is consistent with the close

association of odd-toed ungulates and carnivores, and the

possible assemblage of carnivores and cetartiodactyls

together into superclade Ferungulata (Hou et al. 2009).

Thus, it is possible that bifurcating apical dendrites may be

a feature of animals with large bodies/brains, but to the

general exclusion of primates due to their position within

the euarchontoglire lineage. This raises the interesting

possibility that geometric effects on neocortical neuron

scaling are constrained by phylogenetic history.

Although the arrangement and morphology of cortical

neurons crucially interact with an elephant’s cognitive and

sociobehavioral repertoire, the relationship between corti-

cal architecture and cognition remains unclear. The pres-

ence of V-shaped apical bundles, high synaptic density,

and neurons with a wide, horizontal dendritic spread col-

lectively illustrate that neocortical neurons in elephants are

designed to synthesize a large number of diverse inputs.

Nevertheless, much more research on elephant neocortex is

required before any functional conclusions can be sup-

ported. Certainly, the elephant’s integrative cortical cir-

cuitry seems consistent with its complex spatial and social

abilities, as well as its behaviors indicating empathy and

theory-of-mind. These cortical characteristics may also

contribute to the elephant’s ability to synthesize a variety

of information over an extended period of time. In con-

clusion, although the present study provides an initial

window into the complex neuromorphology of the elephant

cortex, it also highlights the need for more comparative

research to elucidate the multiplicity of evolutionary

options for constructing large, elaborate brains.

Acknowledgments Partial support for this work was provided by the

Thomas M. McKee Professorship in the Natural Sciences (B.J.), The

Colorado College’s divisional research funds (B.J.), the James S. Mc-

Donnell Foundation (grant 22002078, to C.C.S., P.R.H.), and the South

African National Research Foundation (P.R.M.; FA2005033100004).

We would also like to thank Dr. Hilary Madzikanda of the Zimbabwe

Parks and Wildlife Management Authority, and Dr. Bruce Fivaz and the

team at the Malilangwe Trust, Zimbabwe.

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